Without limiting the scope of the invention, its background is described in connection with methods and compositions to modulate blood coagulation.
Antisense oligonucleotides have been used to knockdown protein levels by either translational blocking or splice blocking to control cancer and viral infections with the goal to treat human diseases [1,2]. Knockdown inhibition has been exploited widely in model organisms such as zebrafish, predominantly through use of morpholino oligonucleotides (MOs) to study functions of proteins in both development and disease particularly as a gene discovery tool [3,4]. Typically, these MOs are introduced into the yolks of 1-8-cell-stage zebrafish embryos. Due to the cytoplasmic bridges, it has been thought that MOs rapidly diffuse into these cells allowing ubiquitous cytosolic delivery. However, direct cytosolic delivery of MOs into cells has been difficult to achieve with the exception of microinjections.
Recently, photoactivatable MOs have been introduced to achieve tissue-specific knockdowns in embryos [5]. Another recent development is the conjugation of dendrimeric octaguanidine to MOs (Vivo-MOs) which has resulted in permeability of MOs into cells [6]. Because of this membrane diffusible nature and lack of toxicity, it has been suggested that they might be useful for the use in human therapy [7]. In fact, recently Vivo-MOs have been used to evaluate for use in the treatment of Duchenne Muscular Dystrophy (DMD) [8].
Platelets play a central role in primary hemostasis [9]. During injury, they adhere to subendothelial matrix, become activated, and aggregate to form the primary hemostatic plug. This plug formation involves αIIb or β3 integrin (also known as Glycoprotein IIb/IIIa or Gp IIb/IIIa) activation followed by fibrinogen binding [10,11]. Primary hemostasis in zebrafish also involves thrombocyte aggregation and the fibrinogen receptors as well as thrombocyte functions are all intact in fish [12,13]. These results indicate the technology developed in zebrafish should be translatable to other animal models as well as to humans.
The αIIb or β3 integrin is an adhesion receptor expressed by thrombocytes. This receptor is activated when the thrombocyte is stimulated by ADP, epinephrine, collagen and thrombin. These integrins are essential to the blood coagulation since they have the ability to bind fibrinogen, fibronectin and vitronectin. In patients suffering from Glanzmann's thrombasthenia, an extremely rare coagulopathy, platelets lack glycoprotein IIb/IIIa. Thus, their platelets are less able to adhere to each other and to the underlying tissue of damaged blood vessels. Consequently, no fibrinogen bridging can occur and bleeding time is significantly prolonged.
The αIIb subunit of αIIb integrin is an unparalleled target candidate for inhibition in the primary hemostatic pathway for the following reasons: (i) its inhibition by Vivo-MOs will not cause any non-specific effects of inhibition in other cell types because thrombocytes are the only cells which have αIIb on their membrane surface, (ii) its down regulation could be controlled by the dose of MOs so proper balance of hemostasis could be achieved, (iii) newly generated young thrombocytes will undergo the effect of MOs and the young thrombocytes have previously been shown to initiate thrombus formation at the site of injury, reduction of αIIb in these young thrombocytes could have an immediate effect, (iv) αIIb is the ultimate molecule which receives signals from many pathways and therefore controlled inhibition of this molecule will block the effect of all the pathways, and (vi) it is possible to collect adult thrombocytes, once proof of principle is established for inhibiting thrombocyte function in adult zebrafish, any other candidate factors such as thrombocyte receptors could be targeted for evaluating the function of novel genes involved in hemostatic pathways by studying the biochemical events in thrombocytes.